It is well known that angiogenesis is involved in the pathogenesis of various diseases which include solid tumors, proliferative retinopathy or age-related macular degeneration (AMD) etc associated with ocular neovascularization.
A number of inducing factors such as aFGF, bFGF, TGF-α, TGF-β, HGF, TNF-β, and angiogenin, etc. have been identified in the studies on angiogenesis. Angiogenesis inhibitors may include thrombospondin, a 16 kDa N-terminal fragment of prolactin (Clapp et al., Endocrinology, 133: 1292-1299 (1993)), angiostatin, and endostatin, etc.
Whether angiogenesis is induced or inhibited depends on the balance between angiogenesis inducers and inhibitors (Folkman, J, et al., J. Biol Chem., 267, 10931-10934 (1992)).
Among the angiogenesis inducers, vascular endothelial growth factor (VEGF) is involved in the development and homeostasis of blood vessels and lymphatic vessels, and exhibits significant effects on nerve cells as well. VEGF is produced mainly in vascular endothelial cells, hematopoietic cells and stromal cells under hypoxia or in response to stimulation by cell growth factors such as TGF, Interleukin and PDGF. VEGF binds to a VEGF receptor, and each isoform of VEGF binds to a specific receptor, which induces the formation of homo- or hetero-conjugate of the receptor, to activate each signal transduction pathway (Karsan A., Int J. Mol Med., 5 (5):447-56 (2000)). Signaling specificity of a VEGF receptor is more finely modulated by co-receptors, such as neuropilin, heparan sulfate, integrin, and cadherin, etc. (Zachary I C, et al., Mol. Biol. Cell., 22 (15):2766-76 (2011)). The VEGF is known to be an important mediator of disease-related angiogenesis in tumors and eyes. Also, VEGF mRNA is over-expressed by tumors in the majority of subjects investigated (Berkman et al., J. Clin Invest., 91:153-159 (1993)). Since cancer requires new capillaries as a passage for nutrient supply and waste discharge for its growth, cancer cells and stromal cells thereof secrete VEGF continuously, which is spread throughout tissues and stimulates the migration of vascular endothelial cells (Ferrara. N et al., Nat Rev. Cancer, 2:795-803, (2002)). The neovasculatures induced by cancer cells are characterized by being incomplete as compared to the normally formed capillaries because they are unaided by surrounding cells. Although VEGF binds to receptors VEGFR1, 2 and 3, it is through VEGFR2 that VEGF delivers a signal leading to proliferation, migration and permeability of endothelial cells (H. Zeng et al., J. Biol Chem, 276:26969-26976 (2001)). Therefore, by controlling angiogenesis using drugs targeting VEGF, the proliferation of cancer cells and the diseases associated with angiogenesis can be treated. Among them, antibodies that bind to VEGF can be used as drugs, which undergo a humanization process in order to increase the binding affinity for VEGF and reduce the immunogenicity of the antibodies. Humanized antibodies are described in the literature [Bending, Methods: Comp. Meth. Enzy., 8:83-93 (1995). Anti-VEGF neutralizing antibodies suppress the growth of various human tumor cell lines in nude mouse (Kim et al., Nature, 362:841-844 (1993); Warren et al., etc., J. Clin Invest., 95: 1789-1797 (1995); Borgstroem et al., Cancer Res., 56: 4032-4039 (1996); and Melnyk et al., Cancer Res, 56: 921-924 (1996)), and suppresses intraocular angiogenesis in a model of ischemic vascular disorders of retina (Adamis et al., Arch Ophtalmol., 114:66-71 (1996)). Anti-VEGF antibody may be locally administered into the eye at an effective concentration to decrease the activity of VEGF. Such ischemic retinal disorders may include diabetic retinopathy or age-related macular degeneration.
Anti-VEGF neutralizing antibodies developed so far include bevacizumab (Avastin™, Genentech/Roche) which was approved for colorectal cancer by the FDA in February 2004. Bevacizumab's indication has been extended to the treatment of a total of six types of progressive tumors including metastatic colorectal cancer and progressive ovarian cancer. Additionally, a marketing authorization application for Aflibercept (Bayer Health Care), designed to bind to VEGF, was approved in 2011 by the FDA for the treatment of macular degeneration. However, in 2011 the FDA withdrew breast cancer-related indications of Avastin™ due to its failure to show significant increase in overall survival rates in breast cancer patients compared to placebo. Subsequent reports indicating that Avastin™ increases the risk of heart failure in breast cancer patients suggest that there is a need to improve upon previously developed anti-VEGF neutralizing antibodies, and to determine their exact efficacies at pre-clinical stage. Since Avastin™ does not bind to mouse VEGF, it is difficult to accurately determine its efficacy in pre-clinical models using mice. Thus, an object of the present invention is to develop an antibody that binds to both mouse and human VEGF, thereby acquiring reliability of the results in pre-clinical models; and to increase the binding affinity of the antibody to VEGF, thereby improving anti-tumor effect.
Through biopanning and affinity improvement, the present inventors have developed an antibody comprising a new complementarity determining region (CDR) which has not been previously known, and which through its specific binding to VEGF enables the treatment of tumors and various intraocular neovascular disorders.